The long-term goal of this project is to elucidate the molecular basis for DNA polymerase fidelity and the role of protein conformational dynamics in polymerase function. Accurate nucleotide selection and proofreading by DNA polymerases are essential for proper cellular physiology and the faithful transmission of genetic information during cell division. This project will focus on dynamic conformational changes of the polymerase-DNA complex that are linked to the nucleotide incorporation and 32-52 exonucleolytic proofreading activities. We will study the high fidelity Klenow fragment polymerase, which provides a well-characterized model system. The specific aims are: (1) Develop single-molecule fluorescence methods to monitor the conformational dynamics of DNA polymerases. (2) Establish how functionally important conformational changes of DNA polymerase are linked to selection of a correct incoming nucleotide substrate. (3) Detect DNA polymerase activity at the single-molecule level in real-time. (4) Discover how a DNA primer/template travels between the separate polymerase and 32-52 exonuclease sites during proofreading. (5) Establish how the polymerase and exonuclease activities are physically coordinated to achieve high fidelity DNA replication. Novel single-pair FRET systems will be established to monitor enzyme conformational changes during nucleotide incorporation and translocation of DNA during proofreading. These new tools will open a unique window into the inner workings of DNA polymerase enzymes and will be broadly applicable to other protein-DNA complexes.